CN117844811A - sgRNA composition for targeted knockout of CD70 gene and application thereof - Google Patents
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Abstract
The invention relates to an sgRNA composition for targeting knockout CD70 gene and application thereof, wherein the sgRNA composition comprises three sgRNAs with targeting CD70 and targeting sequences of SEQ ID NO. 1-3 respectively. The invention also provides a method for carrying out gene editing on the intracellular CD70 gene by using the sgRNA composition and a CD70 gene knockout cell obtained by construction. The sgRNA composition provided by the invention can efficiently guide Cas nuclease to bind with a target sequence of a targeted CD70 gene, has the advantage of high knockout efficiency, can efficiently construct a cell line which does not express the CD70 gene, and is beneficial to developing in vitro function experiments of CD70 CAR-T cells.
Description
Technical Field
The invention relates to the field of gene editing, in particular to a targeting CD70 gene knockout sgRNA composition and application thereof.
Background
CRISPR-Cas9 is a third generation gene editing technology which is introduced by a ZFN, TALENs and other gene editing technologies, is one of the technologies with highest efficiency, simplest and lowest cost in the existing gene editing and gene modification, and is the most mainstream gene editing system at present. The genome screening function of the CRISPRP-Cas 9 system has the advantages of high specificity and irreversibility, and is widely applied to genome screening. The technology recognizes a target genome sequence through an artificially designed sgRNA (guide RNA), and guides Cas9 protease to effectively cut a DNA double chain so as to form double chain break. In general, cells repair fragmented DNA using efficient non-homologous end joining (NHEJ). However, in the repair process, a mismatch phenomenon of base insertion or deletion usually occurs, which causes frame shift mutation, and the target gene is disabled, so that gene knockout is realized.
Kidney cancer, also called renal cell carcinoma, is a tumor with a high malignancy in the urinary system, and is one of the most common tumors, and it accounts for 2% -3% of adult malignant tumors and about 20% of pediatric malignant tumors in urogenital system tumors in China. The treatment methods of kidney cancer mainly comprise surgical excision, targeted therapeutic drugs and immunotherapy. CD70 is currently considered a novel specific tumor marker for kidney cancer. Thus, CD70 is a good target for tumor targeting and immunotherapy. Currently, a variety of CD 70-targeting drugs have been developed, including CAR-T cells, monoclonal antibodies, ADCs, and the like.
CD70 is a type II transmembrane protein, which is expressed predominantly in activated T cells, B cells and mature dendritic cells. The CD70 receptor is CD27, and the action of CD70 with CD27 can promote activation, proliferation and differentiation of T cells and B cells, and modulate immune responses. Under normal conditions, the expression of the antigen is highly expressed on the surfaces of activated T cells and B cells, and the expression of the antigen is down-regulated along with the reduction of antigen stimulation in the late stage of immune response, so that the expression time is short. However, in pathological conditions, CD70 is highly expressed in a variety of tumor cells, which bind to the T cell receptor CD27 by expressing CD70, and chronic co-stimulation results in T cells expressing immune checkpoints such as PD-1, tim-3, etc., leading to immune function depletion. The high expression of CD70 in tumors may suggest that tumors utilize CD70 to control tumor infiltrating lymphocytes that express CD27, thereby generating immune evasions. Abnormal expression of CD70 is significantly associated with the development of tumors and poor prognosis in patients. CD70 has high expression level and high positive detection rate in various malignant tumors, especially kidney cancer, and has become a potential target for treating kidney cancer with both effectiveness and safety. CAR-T cell therapy against CD70 as a target is a hotspot for renal cancer therapy.
According to the query ClinicalTrials, there are many studies related to CD70 CAR-T, such as Zhejiang university and Yake Biotechnology, shenzhen Geno-Immune Medical Institute, CRISPR Therapeutics, allogene Therapeutics. The indications are mainly renal cell carcinoma, and other blood tumors such as acute myelogenous leukemia non-Hodgkin lymphoma, multiple myeloma and other B cell malignant tumors are included. No relevant products targeting CD70 are currently marketed, but some studies have entered the clinical stage, with major indications being hematological tumors and renal cancers. According to the Chinese national drug administration drug review Center (CDE) and the United states Food and Drug Administration (FDA), three CD70 CAR-T cell therapy products are currently available worldwide for IND approval into registered clinics to explore treatment of solid tumors.
CD 70-targeted CAR-T cell therapy products require evaluation of the antitumor activity of CD70 CAR-T cells of different origins on kidney cancer cell lines and mouse tumor models when performing in vivo and in vitro functional assays. A common kidney cancer cell line is ACHN cells, which are derived from pleural effusion of a 22 year old white man suffering from renal cell adenocarcinoma, and are immortalized cells formed by human embryonic kidney cells transfected with sheared human adenovirus 5 (Ad 5). In vivo studies, ACHN cells are often used to construct a mouse kidney cancer model for research in exploring renal cancer treatment methods. CD70 is highly expressed in ACHN, can be identified by CD70 CAR-T cell targeting, and is an ideal kidney cancer cell line in vitro function verification. In assessing the specific antitumor activity of CD70 CAR-T cells against ACHN cells, we need to verify from both the front and back sides, both to demonstrate the killing effect of CD70 CAR-T cells against ACHN cells, and to demonstrate that the killing effect of CD70 CAR-T cells against ACHN cells will no longer exist when CD70 is no longer expressed in ACHN cells. Thus, a CD70 knockout ACHN cell line was established. The prior art discloses some sgrnas targeting knockout CD70, and no sgRNA composition products capable of efficiently knocking out the ACHN cell CD70 gene as disclosed in the present application are currently known.
Disclosure of Invention
The invention provides a sgRNA composition for targeted knockout of CD70 gene and application thereof.
In a first aspect, the invention provides an sgRNA composition for gene editing of intracellular CD70 genes, said sgRNA composition comprising three sgRNAs targeting CD70 with targeting sequences of SEQ ID NO 1-3, respectively.
In one or more embodiments, the sgRNA composition comprises three sgRNAs as shown in SEQ ID NOS.4-6.
In one or more embodiments, the mole percent of the three sgrnas shown in SEQ ID NOs 4-6 in the sgRNA composition is (0.000001% -99.999999%); in some embodiments, the mole percent is (40% -60%); in some embodiments, the mole percent is 50%:50%:50%.
In one or more embodiments, the sgrnas are modified. Preferably, the modification is one or more of nucleotide modification, internucleotide modification, 5 'terminal modification and 3' terminal modification, sugar-internucleotide linkage modification. Preferably, the modification is one or more of methylation modification, methoxy modification, fluorination modification or thio modification.
In a second aspect, the present invention provides a composition for gene editing of a CD70 gene in a cell, comprising:
(i) Three sgrnas in the sgRNA composition described in any of the embodiments herein; or, a nucleic acid encoding three sgrnas in a sgRNA composition as described in any of the embodiments herein; and
(ii) A protein comprising a leader nucleotide sequence-a programmable DNA binding protein domain (guide nucleotide sequence-programmable DNA binding protein domain); or, a nucleic acid encoding said protein comprising a leader nucleotide sequence-programmable DNA binding protein domain.
In one or more embodiments, the guide nucleotide sequence-programmable DNA binding protein domain is derived from a Cas nuclease. Preferably, the nuclease is selected from one or more of Cas9, cas3, cas8a, cas8b, cas10d, cse1, csy1, csn2, cas4, cas10, csm2, cmr5, fok1, cpf 1. More preferably, the Cas9 is selected from Cas9 derived from streptococcus pneumoniae, streptococcus pyogenes or streptococcus thermophilus.
In a third aspect, the present invention provides a method of gene editing of a CD70 gene in a cell, the method comprising the steps of: introducing into a cell a protein comprising a leader nucleotide sequence-programmable DNA binding protein domain and an sgRNA composition as described in any of the embodiments herein that directs cleavage of the CD70 gene by the protein comprising the leader nucleotide sequence-programmable DNA binding protein domain and formation of a cleavage site, and editing the CD70 gene.
In one or more embodiments, the guide nucleotide sequence-programmable DNA binding protein domain is derived from a Cas nuclease. Preferably, the nuclease is selected from one or more of Cas9, cas3, cas8a, cas8b, cas10d, cse1, csy1, csn2, cas4, cas10, csm2, cmr5, fok1, cpf 1. More preferably, the Cas9 is selected from Cas9 derived from streptococcus pneumoniae, streptococcus pyogenes or streptococcus thermophilus.
In one or more embodiments, the means for introducing the protein comprising the leader nucleotide sequence-programmable DNA binding protein domain and the sgRNA composition into the cell is selected from electroporation, vector transformation, transfection, heat shock, transduction, gene gun, or microinjection. Preferably, electroporation is used.
In a fourth aspect, the invention provides a gene-edited cell prepared by a method as described in any of the embodiments herein.
In a fifth aspect, the invention provides a sgRNA composition as described in any of the embodiments herein, the use of a composition as described in any of the embodiments herein for the preparation of a product for gene editing of intracellular CD 70.
The invention has the beneficial effects that: the sgRNA composition provided by the invention can efficiently guide Cas nuclease to bind with a target sequence of a targeted CD70 gene, has the advantage of high knockout efficiency, can efficiently construct a cell line which does not express the CD70 gene, and is beneficial to developing in vitro function experiments of CD70 CAR-T cells.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1: schematic representation of human CD70 genome information. The CD70 gene of human origin contains only three exons, the first one containing the 5' UTR sequence and the 161bp coding sequence. We designed three sgRNAs in the coding region of the first exon.
Fig. 2: three of the sgrnas have sequence information.
Fig. 3: the knockdown efficiency of ACHN intracellular CD70 was flow tested (three sgRNA mixes). In electrotransformation, three sgrnas were mixed and electrotransformed. The results of the flow test for CD70 expression indicate that the CD70 knockout group hardly detects CD70 expression and the knockout efficiency is 100% compared with the wild type control group.
Fig. 4: the efficiency of the knockdown of CD70 (single sgRNA) in ACHN cells was flow tested. Three sgrnas were electrotransferred separately during electrotransfer. The results of the flow detection of the expression of CD70 show that compared with a wild type control group, the expression of CD70 in the electric conversion sgRNA1 group is the lowest, the knocking-out efficiency is the highest, and the knocking-out efficiency is about 87%.
Fig. 5: the expression of CD70 in ACHN (CD 70 KO) monoclone was detected by flow. The results of the flow test on the expression of CD70 in ACHN (CD 70 KO) monoclone show that neither 1A3 nor 1A5 monoclone expresses CD70, which indicates that the construction of CD70 knockout ACHN cell monoclone is successful.
Description of the embodiments
Through extensive and intensive studies, the inventors of the present application selected several sgrnas, which were, on the one hand, electrotransferred into ACHN cells separately, and, on the other hand, mixed together, and then electrotransferred into ACHN cells. Subsequent analyses at the protein level all compared the knockdown efficiency of CD70 in ACHN cells, aimed at selecting the experimental group with the highest knockdown efficiency of CD70 knockdown, facilitating the later faster preparation of CD70 knockdown ACHN cell monoclonal. The method has positive significance for the later in-vitro functional experiment verification and evaluation of the specific anti-tumor activity of the CD70 CAR-T cells on ACHN cells.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and other references mentioned herein are incorporated by reference.
As used herein, the terms "comprising," having, "or" including "include" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … ….
CRISPR/Cas gene editing technology
The terms "CRISPR/Cas technology", "CRISPR/Cas genome editing technology", "CRISPR/Cas genome editing method" as used herein refer generally to a technology for modifying a DNA sequence of interest using a CRISPR/Cas system. The "CRISPR/Cas technology" may also comprise methods for gene expression regulation using similar principles, such as CRISPR/dCas9 based gene expression regulation technologies.
sgRNA compositions
As used herein, "sgRNA," "guide RNA," or "sgRNA of the invention" are used interchangeably and refer to a sgRNA that targets the CD70 gene. The sgRNA composition comprises three sgRNAs with targeting sequences of SEQ ID NO 1-3 respectively for targeting CD 70. In some embodiments, the sgRNA composition comprises three sgRNAs as shown in SEQ ID NOs 4-6.
In some embodiments, any one or several of the sgrnas in the sgRNA composition are modified. The allowable modification is one or more of nucleotide modification, internucleotide modification, 5 'terminal modification, 3' terminal modification, sugar modification and sugar-internucleotide linkage modification. The type of base modification allowed is one or more of methylation modification, methoxy modification, fluorination modification or thio modification. In preferred embodiments, the sgrnas comprise chemical modifications of any one, any several or any consecutive several bases of the 1 st to n th bases of the 5 'end, and/or chemical modifications of any one, any several or any consecutive several bases of the 1 st to n th bases of the 3' end; the n is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably, the sgrnas comprise chemical modifications of one, two, three, four or five bases at the 5 'end, and/or chemical modifications of one, two, three, four or five bases at the 3' end. For example, the 1 st, 2 nd, 3 rd, 4 th, 5 th or 1-2 nd, 1-3 rd, 1-4 th, 1-5 th base of the 5' end of the sgRNA is chemically modified; and/or, carrying out chemical modification on the 1 st base, the 2 nd base, the 3 rd base, the 4 th base, the 5 th base or the 1 st to 2 nd bases, the 1 st to 3 rd bases, the 1 st to 4 th bases and the 1 st to 5 th bases of the 3' end of the sgRNA. In a specific embodiment of the invention, both the 5 'and 3' ends of the sgrnas bear three thio modifications.
Composition and method for producing the same
The composition for gene editing of intracellular CD70 gene of the present invention comprises: (i) Three sgrnas in the sgRNA composition described in any of the embodiments herein; or, a nucleic acid encoding three sgrnas in a sgRNA composition as described in any of the embodiments herein; and (ii) a protein comprising a leader nucleotide sequence-a programmable DNA binding protein domain; or, a nucleic acid encoding said protein comprising a leader nucleotide sequence-programmable DNA binding protein domain.
In some embodiments, the guide nucleotide sequence-programmable DNA binding protein domain is derived from a Cas nuclease. Optionally, the nuclease is selected from one or more of Cas9, cas3, cas8a, cas8b, cas10d, cse1, csy1, csn2, cas4, cas10, csm2, cmr5, fok1, cpf 1. Optionally, the Cas9 is selected from Cas9 derived from streptococcus pneumoniae, streptococcus pyogenes, or streptococcus thermophilus. Herein, originating from streptococcus pneumoniae, streptococcus pyogenes or streptococcus thermophilus is understood to be Cas9 originating from or derived from streptococcus pneumoniae, streptococcus pyogenes or streptococcus thermophilus. By derivatized enzyme is meant an enzyme that has a high degree of sequence homology to the wild-type enzyme, e.g., has been mutated or modified at certain positions.
Nuclease refers to a nuclease that includes one or more DNA binding domains and one or more DNA cleavage domains. The nucleases of the present invention may be designed and/or modified from naturally occurring nucleases or from artificially engineered nucleases. Nucleases of the invention include homing endonucleases, megaTALs, transcription activator-like effector nucleases (TALENs), zinc Finger Nucleases (ZFNs), and clustered regularly interspaced short palindromic repeats CRISPR/Cas nuclease systems. In a preferred embodiment, the nucleases of the present invention comprise a CRISPR/Cas nuclease system.
In the present invention, the CRISPR/Cas nuclease system comprises a Cas nuclease and one or more RNAs that recruit the Cas nuclease to a target site, such as transactivation cRNA (tracrRNA) and CRISPR RNA (crrnas) or single guide RNAs (sgrnas). crrnas and tracrrnas may be designed with a polynucleotide sequence of "single guide RNA" or "sgRNA".
Method
The method for gene editing of intracellular CD70 gene of the present invention comprises the steps of: introducing into a cell a protein comprising a leader nucleotide sequence-programmable DNA binding protein domain and an sgRNA that directs cleavage of the CD70 gene by the protein comprising the leader nucleotide sequence-programmable DNA binding protein domain and formation of a cleavage site, and editing the CD70 gene; the sgrnas are three sgrnas in the sgRNA compositions described in any of the embodiments herein.
In some embodiments, the guide nucleotide sequence-programmable DNA binding protein domain is derived from a Cas nuclease. Optionally, the nuclease is selected from one or more of Cas9, cas3, cas8a, cas8b, cas10d, cse1, csy1, csn2, cas4, cas10, csm2, cmr5, fok1, cpf 1. Optionally, the Cas9 is selected from Cas9 derived from streptococcus pneumoniae, streptococcus pyogenes, or streptococcus thermophilus. Herein, originating from streptococcus pneumoniae, streptococcus pyogenes or streptococcus thermophilus is understood to be Cas9 originating from or derived from streptococcus pneumoniae, streptococcus pyogenes or streptococcus thermophilus. By derivatized enzyme is meant an enzyme that has a high degree of sequence homology to the wild-type enzyme, e.g., has been mutated or modified at certain positions.
In some embodiments, the means for introducing the protein comprising the leader nucleotide sequence-programmable DNA binding protein domain and the sgRNA into the cell is selected from electroporation, vector transformation, transfection, heat shock, transduction, gene gun or microinjection, which is not limited by the present invention, e.g., nuclease and sgRNA can be formed into a complex, which is then introduced into the cell by electroporation.
However, the sgRNA-mediated gene editing system of the present invention has superior genome editing capabilities compared to the prior art. In a specific embodiment, the gene inactivation rate obtained using the gene editing system of the present invention is higher than 90%, more preferably higher than 95%, and still more preferably up to 100%.
Cells
The CD70 knockout cell can be constructed using the method of gene editing of the intracellular CD70 gene described in any of the embodiments herein. The CD70 gene knockout means that the CD70 expression amount of the cell is reduced compared to a wild-type cell; preferably, the decrease is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more than 90%; more preferably, the decrease is 95%, 96%, 97%, 98% or 99% or more.
In some embodiments, the cell is a tumor cell, such as a renal cell carcinoma, acute myelogenous leukemia, non-hodgkin lymphoma, multiple myeloma, mantle cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, pancreatic cancer, breast cancer, or glioblastoma cell, such as, in particular, an ACHN cell line, 786-O cell line, a498 cell line, caki-2 cell line, caki-1 cell line, caki-2 cell line, L99-15 cell line, CCRF-CEM cell line, HL-60 cell line, THP-1 line, U937 cell line, jeko-1 cell line, raji cell line, MM. S cell line, H929 cell line, U266 cell line, RPMI8226 cell line, jeko-1 cell line, grante519 line, mino cell line, Z138 cell line, RF-1 cell line, recledo cell line, tooci line, cfi-10 line, cfly-pac-7, MDA-172, MDA-87, or MCF-172 cell line, MDA-7, or a-172 cell line.
Use of the same
The sgRNA compositions of the invention can be prepared as products, such as kits, for gene editing of intracellular CD 70. The kit may further comprise nucleases and the like. The method for gene editing by using the sgRNA composition comprises the following steps: introducing a protein comprising a leader nucleotide sequence-programmable DNA binding protein domain and an sgRNA that directs cleavage of the CD70 gene by the protein comprising the leader nucleotide sequence-programmable DNA binding protein domain and formation of a cleavage site into cells, and editing the CD70 gene. The means for introducing the protein comprising the leader nucleotide sequence, the programmable DNA binding protein domain, and the sgRNA into the cell is selected from electroporation, vector transformation, transfection, heat shock, transduction, gene gun or microinjection, which is not particularly limited by the present invention, and for example, the nuclease and the sgRNA may be formed into a complex, and the complex is introduced into the cell by electroporation.
The invention will be illustrated by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The methods and materials used in the examples are those conventional in the art, unless otherwise indicated.
Examples
1. Experimental method
Design and Synthesis of sgRNA
To knock out CD70 in ACHN cells (source: national academy of sciences cell bank), we used CRISPR-Cas9 gene editing technology to implement. We designed several sgRNA sequences and selected for synthesis and modification by aurora: 2' -O-methyl and phosphorothioate modifications at the first three 5' and 3' terminal RNA residue (translated herein as three thio modifications at both the 5' and 3' ends) (FIGS. 1 and 2).
2. Electric rotating device
We selected the Lonza electrotransport apparatus to electrotransport deliver Cas9 protein (manufacturer: genScript; cat# 203702-500) and sgRNA as RNP complexes into ACHN cells for gene editing. Preparation of sgRNA working solution and electrotransfer buffer is required prior to electrotransfer. Preparation of sgRNA working solution: the synthesized 3nmol of EasyEdit sgRNA in lyophilized powder form was dissolved in RNase-/DNase-free and pyrogen-free water at a final concentration of 100. Mu.M (100 pmol/. Mu.l). Preparation of electrotransport buffer: SF electrotransfer solution was mixed with a supplementary solution (kit: SF Cell Line 4D-Nucleofector X Kit S; manufacturer: lonza; product number: V4 XC-2032) in a ratio of 4.5:1 to prepare electrotransfer buffer, and equilibrated to room temperature after mixing. ACHN cells were counted for digestion and the desired amount of cells (2E 5/20ul reaction) was collected by centrifugation. The following reagents were added sequentially in a 1.5ml Ep tube of sterile, DNase/RNase Free: 5ul of mixed electrotransport buffer, 1.2ul (120 pmol) of sgRNA (0.4 ul (40 pmol) of each sgRNA in case of electrotransport three sgRNAs), 1.6ul (40 pmol) of Cas9 protein were added to each reaction system, and after thoroughly mixing, incubated for 10 minutes at room temperature to form RNP mixture. The centrifuged cell pellet was resuspended in 15ul of electrotransfer buffer and the cell suspension was then added to a 1.5ml Ep tube containing RNP and mixed well and transferred to a 16 well Nucleocuvette electrotransfer strip. The 4D-nucleofector (tm) electrotransport meter is started, the appropriate electrotransport program CM130 is selected, the electrotransport strip is placed in the strip bracket of 4D-NucleofectorTM X Unit, and the electrotransport process is started. Immediately after the run was completed, the Nucleocuvette (TM) electrotransport strip was carefully removed from the machine strip tray, 80ul of pre-warmed R10 medium was added to each well and placed in a 37℃incubator for 30min. The electrotransport plate was removed and the cells transferred to a 48-well plate pre-plated with 300ul of R10 medium and placed in a 37℃incubator for culture. The knockout efficiency of CD70 is judged by a flow detection mode after 3-7 days of culture.
3. Flow detection of knockout efficiency of ACHN intracellular CD70
Culturing the cells after electric transformation for 3-7 days, taking ACHN cells of wild type and CD70 knockout groups respectively, washing the cells once by PBS, and staining the cells by using a flow antibody PE anti-human CD70 (manufacturer: biolegend; product number: 355104) and Zombie red for 30min by a 4-degree refrigerator. After staining, the cells were washed once with PBS, resuspended in 100ul PBS, and the cells were placed in flow cytometer SA3800 for flow detection. The protein expression of CD70 in each group was analyzed by Flowjo software after the end of the flow, and the knockout efficiency of CD70 in the CD70 knockout group was analyzed based on the expression of CD70 in wild-type ACHN cells.
4. Picking monoclonal to verify CD70 knockout
After flow assay confirmed CD70 gene knockout in ACHN cells, we needed to prepare a monoclonal for uniformity of subsequent experiments. We used limiting dilution to dilute the ACHN mixed cell suspension with CD70 knockdown, and then spread the cell suspension into 96-well plates, 100ul of system per well, plated to ensure 0.5 cells per well as standard. And after the proliferation and amplification of the monoclonal cells to a certain cell number, detecting the expression condition of CD70 in ACHN in the monoclonal cells in a flow mode.
2. Experimental results
The flow detection result shows that three sgrnas are mixed and then subjected to electrotransformation, and compared with a wild type control group, the CD70 knockout group can hardly detect the expression of CD70, and the knockout efficiency is 100 percent (figure 3); the three sgrnas were electrotransformed, respectively, and compared with the wild-type control group, the electrotransformed sgrnas 1 group had the lowest CD70 expression and the highest knockout efficiency, which was about 87% (fig. 4). Monoclonal cells were selected to verify CD70 expression, and none of the resulting monoclonal expressed CD70, indicating successful construction of a CD70 knockout ACHN cell monoclonal (fig. 5).
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An sgRNA composition for gene editing of intracellular CD70 gene, wherein the sgRNA composition comprises three sgrnas targeting CD70 with targeting sequences of SEQ ID NOs 1 to 3, respectively; preferably, the sgRNA composition comprises three sgRNAs as shown in SEQ ID NOS.4-6.
2. The sgRNA composition of claim 1, wherein the three sgrnas shown in SEQ ID NOs 4-6 in the sgRNA composition are in mole percent (0.000001% -99.999999%); preferably, the mole percentage is (40% -60%); more preferably, the mole percent is 50% to 50%.
3. The sgRNA composition of claim 1, wherein any one or several of the sgrnas in the sgRNA composition are modified; preferably, the modification is one or more of nucleotide modification, internucleotide modification, 5 'terminal modification and 3' terminal modification, sugar-internucleotide linkage modification; preferably, the modification is one or more of methylation modification, methoxy modification, fluorination modification or thio modification.
4. A composition for gene editing of intracellular CD70 gene comprising:
(i) Three sgrnas in the sgRNA composition of any one of claims 1 to 3; or, a nucleic acid comprising three sgrnas in the sgRNA composition of any one of claims 1 to 3; and
(ii) A protein comprising a leader nucleotide sequence-a programmable DNA binding protein domain; or, a nucleic acid encoding said protein comprising a leader nucleotide sequence-programmable DNA binding protein domain.
5. The composition of claim 4, wherein the guide nucleotide sequence-programmable DNA binding protein domain is derived from a Cas nuclease; preferably, the nuclease is selected from one or more of Cas9, cas3, cas8a, cas8b, cas10d, cse1, csy1, csn2, cas4, cas10, csm2, cmr5, fok1, cpf 1; more preferably, the Cas9 is selected from Cas9 derived from streptococcus pneumoniae, streptococcus pyogenes or streptococcus thermophilus.
6. A method of gene editing of an intracellular CD70 gene, the method comprising the steps of: introducing into a cell a protein comprising a leader nucleotide sequence-programmable DNA binding protein domain and an sgRNA composition according to any of claims 1 to 3, which directs the protein comprising a leader nucleotide sequence-programmable DNA binding protein domain to cleave the CD70 gene and form a cleavage site, to edit the CD70 gene.
7. The method of claim 6, wherein the guide nucleotide sequence-programmable DNA binding protein domain is derived from a Cas nuclease; preferably, the nuclease is selected from one or more of Cas9, cas3, cas8a, cas8b, cas10d, cse1, csy1, csn2, cas4, cas10, csm2, cmr5, fok1, cpf 1; more preferably, the Cas9 is selected from Cas9 derived from streptococcus pneumoniae, streptococcus pyogenes or streptococcus thermophilus.
8. The method of claim 6, wherein introducing the protein comprising the leader nucleotide sequence-programmable DNA binding protein domain and the sgRNA composition into the cell is selected from electroporation, vector transformation, transfection, heat shock, transduction, gene gun, or microinjection; preferably, electroporation is used.
9. A genetically edited cell prepared by the method of any one of claims 6-8.
10. Use of the sgRNA composition of any one of claims 1 to 3, the composition of any one of claims 4 to 5 for the preparation of a product for gene editing of intracellular CD 70.
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